Intermittent dosing of mdm2 inhibitor

ABSTRACT

The present disclosure relates to mdm2 inhibitors for use in specific dosing schedules. It was found that if sufficiently potent or, in alternative, sufficiently high dose of a Mdm2 inhibitor is used, it can cause antineoplastic effect by triggering much longer lasting antiproliferative mechanism in cells. The long lasting effect can sustain for several weeks after a single dose, which eliminates the need for daily treatment and allows administering the Mdm2i intermittently. A treatment with the intermittent dosing schedule of a Mdm2 inhibitor can be combined with a daily treatment of the Mdm2i or with another pharmaceutically acceptable ingredient.

RELATED APPLICATIONS

The present disclosure is a continuation of US Patent Application Ser.No. (USPAN) 16/898,886 filed Jun. 11, 2020, which is a continuation ofUS Patent Application Ser. No. (USPAN) 16/221,650 filed Dec. 17, 2018,which is a continuation of USPAN Ser. No. 15/849,770 filed Dec. 21,2017, which is a continuation of USPAN Ser. No. 15/321,042 filed Dec.21, 2016, which is a 371 of International Application PCT/IB2015/054792filed Jun. 25, 2015 which claims priority to U.S. ProvisionalApplication No. 62/017,406 filed Jun. 26, 2014 which is incorporatedherein in its entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates to mdm2 inhibitors for use in specificdosing schedules.

BACKGROUND OF THE DISCLOSURE

The protein p53 is a transcription factor that controls the expressionof a multitude of target genes involved in DNA damage repair, apoptosisand cell cycle arrest, which are all important phenomena counteractingthe malignant growth of tumors. p53 is thus critical for maintaininggenetic stability and preventing tumor development. The TP53 gene is oneof the most frequently mutated genes in human cancers. It is reportedthat approximately half of all cancers have inactivated p53, caused bydirect mutation. In cancers in which the p53 gene is not mutated,functional inactivation at the protein level has been demonstrated. Oneof the mechanisms of p53 inactivation described is through itsinteraction with human homolog of MDM2 (Mouse double minute 2). Mdm2 istherefore an important negative regulator of the p53 tumor suppressor.Mdm2 protein functions both as an E3 ubiquitin ligase, that leads toproteasomal degradation of p53, and an inhibitor of p53 transcriptionalactivation. Often Mdm2 is found amplified in p53 wild-type tumors.

Mdm2 inhibitors have been developed that inhibit p53-mdm2 interactionand can elicit antineoplastic effect.

US2013/0245089 disclosed a method of treating a patient suffering withcancer by administering to the patient 4-{[(2R,35,4R,5S)-4-(4-Chloro-2-fluoro-phenyl)-3-(3-chlorofluoro-phenyl)-4-cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxy-benzoic acidin an amount of from about 800 to about 3000 mg/day for anadministration period of up to about 7 days, on days 1-7, of a 28 daytreatment cycle, followed by a rest period of from about 21 to about 23days.

A paper in Clinical Cancer Research by B. Higgins et al. (May 2014)disclosed a 28-day cycle schedule, where RG7388 is administered onceweekly three times followed by 13 days of rest (28-day cycle schedule),or where the drug is administered for 5 consecutive days of a 28-dayschedule.

Mdm2 inhibitors and how to prepare them were disclosed for example inWO2013111105 or WO2011076786.

SUMMARY OF THE DISCLOSURE

It has been unexpectedly discovered that an advantageous dosing regimenfor a Mdm2 inhibitor (hereinafter “Mdm2i”) can be designed byunderstanding the biology of the drug target and how the Mdm2iconcentration can alter signaling of the downstream pathway to affectanti-tumor efficacy and tolerability. Surprisingly, it was found that ifa sufficiently potent Mdm2i or, in alternative, a sufficiently high doseof a Mdm2i is used, it can cause antineoplastic effect by triggeringmuch longer lasting antiproliferative mechanism in cells. When a cancercell is exposed to sufficiently high concentration of the respectiveMdm2i for as short as 8 hours (and proportionally longer if a lowerconcentration is used), Mdm2i causes p21 and Puma mRNA expression tospike within the next 48 to 72 hours, leading to significant inductionof caspase 3/7 activity and thus to substantial apoptosis. In animalsthat have had cancer cells implanted subcutaneously the same effectafter treating the animal with a sufficiently high single dose wasobserved. This led to substantial tumor shrinkage. None of this wasdetected when the Mdm2i exposure was below a certain threshold belowwhich this second modality of Mdm2i was not activated. The knowledge ofthe second modality of Mdm2i can help plan clinical trials in a way toreduce side effects due to an on-target effect of the drug.

Interestingly, it was observed that a long lasting effect can besustained for several weeks after a single dose, which eliminates theneed for daily treatment and allows administering the drugintermittently. During the breaks with no administration of a drug anorganism can recover from potential on-target effects or side effects;particularly numbers of white blood cells (WBC), neutrophils andplatelets can recover. Administering the Mdm2i at doses that trigger thelong lasting effect causes the Mdm2i to be at least as effective as whendosed daily at lower doses, and can be better tolerated. Less frequentdosing can also lead to better patient friendliness, patient compliance,and particularly where the drug is administered intravenously, can havesignificant patient benefits. For example, the local injection siteirritations can properly heal before the next dose is due.

The intermittent dosing of an Mdm2i with a sustained effect can becombined with a dosing regimen comprising a daily administration of alower dose compared to the dose used to achieve a sustained effect. Thecombination of intermittent dosing of a first dose and daily dosing of asecond dose yields synergistic effect in terms of the compound efficacy,which is observed for example as a tumor shrinkage or tumor regression.In addition, due to better tolerability of Mdm2i when administeredintermittently, the drug can be used in combination with otherantineoplastic agents. The combination of a Mdm2i and anotherantineoplastic agent can exploit improved tolerability of the Mdm2i whenit is dosed intermittently, while increasing the overall efficacy of thecombination therapy with a second antineoplastic agent.

Specifically, the present disclosure provides the following aspects,advantageous features and specific embodiments, respectively alone or incombination, as listed in the following items:

1. A MDM2i for use in the treatment of cancer, wherein MDM2i isadministered to a subject intermittently in at least three consecutivedoses and the period between each two consecutive doses is at least 2weeks.

2. The MDM2i for use in the treatment of cancer according to item 1,wherein MDM2i is administered to a subject intermittently and the periodbetween each two consecutive administrations is at least 3 weeks and notlonger than 60 days.

3. The MDM2i for use in the treatment of cancer according to item 1 or2, wherein MDM2i is administered to a subject intermittently and theperiod between consecutive administrations is 3 weeks.

4. The MDM2i for use in the treatment of cancer according to any one ofitems 1 to 3, wherein MDM2i is administered intravenously.

5. A MDM2i for use in the treatment of cancer, wherein MDM2i isadministered to a subject in a first and a second dose and the firstdose is administered on the same day as the second dose, consecutivedays or a different day to the second dose, wherein two consecutiveadministrations of the first dose are administered intermittently atleast every 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks or every 60 days,and not longer than every 60 days, and the first and the second dose arenot the same.

6. The MDM2i for use in the treatment of cancer according to item 5,wherein the second dose is administered daily, optionally with a break.

7. The MDM2i for use in the treatment of cancer according to item 6,wherein the break is at least 1 day long, 2 days, 3 days, 4 days, 1week, 2 weeks, or 3 weeks and at most 26 days long.

8. The MDM2i for use in the treatment of cancer according to any one ofitems 5 to 7, wherein the second dose is administered 1 to 14 days afterthe first dose has been administered.

9. The MDM2i for use in the treatment of cancer according to any one ofitems 5 to 8, wherein the second dose is administered for two weeksfollowed by a period of two weeks without treatment and then repeatingthe cycle.

10. The MDM2i for use in the treatment of cancer according to any one ofitems 5 to 9, wherein the first dose is higher than the second dose.

11. The MDM2i for use in the treatment of cancer according to any one ofitems 5 to 10, wherein at least one of the first or the second dose isadministered intravenously.

12. The MDM2i for use in the treatment of cancer according to any one ofitems 5 to 11, wherein two consecutive administrations of the first doseare administered intermittently at least every 2 weeks.

13. The MDM2i for use in the treatment of cancer according to any one ofitems 5 to 11, wherein two consecutive administrations of the first doseare administered intermittently at least every 3 weeks.

14. The MDM2i for use in the treatment of cancer according to any one ofitems 1 to 13, wherein the cancer is bladder, breast, brain, head andneck, liver, oral, biliary tract, acute and chronic lymphoid leukemia,acute and chronic myeloid leukemia, chronic myelomonocytic leukemia,colorectal, gastric, gastrointestinal stromal, hepatocellular, glioma,lymphoma, melanoma, multiple myeloma, myeloproliferative disease,neuroendocrine, lung, non-small cell lung, pancreatic, ovarian,prostate, renal cell, sarcoma, liposarcoma and thyroid cancer.

15. The MDM2i for use in the treatment of cancer according to any one ofitems 1 to 13, wherein the cancer is melanoma, lung cancer orneuroblastoma.

16. The MDM2i for use in the treatment of cancer according to any one ofitems 1 to 13, wherein the cancer is melanoma.

17. Use of a MDM2i for the preparation of a medicament for the treatmentof a cancer, wherein MDM2i is administered intermittently in at leastthree consecutive doses and the period between each two consecutivedoses is at least 2 weeks, at least 3 weeks, at least 4 weeks, at least6 weeks or 60 days, and not longer than 60 days.

18. A method of treating cancer, wherein MDM2i is administered to asubject in need thereof intermittently in at least three consecutivedoses and the period between each two consecutive doses is at least 2weeks, 3 weeks, at least 4 weeks, at least 6 weeks or 60 days, and notlonger than 60 days.

19. The MDM2i for use in the treatment of cancer according to any one ofitems 1 to 16, the use of a MDM2i for the preparation of a medicamentfor the treatment of a cancer according to item 17, or the method oftreating cancer according to item 18, wherein MDM2i is administered to ahuman.

20. The MDM2i for use in the treatment of cancer according to any one ofitems 1 to 16, or 19, the use of a MDM2i for the preparation of amedicament for the treatment of a cancer according to item 17 or 19, orthe method of treating cancer according to item 18 or 19, wherein theMDM2i is selected from the group consisting of:

(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one

(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one

(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(6-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-pyridin-3-yl)-1,4-dihydro-2H-isoquinolin-3-one

(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(6-{methyl-[4-(3-methyl-4-oxo-imidazolidin-1-yl)-trans-cyclohexylmethyl]-amino}-pyridin-3-yl)-1,4-dihydro-2H-isoquinolin-3-one

(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(5-{methyl-[4-(3-methyl-4-oxo-imidazolidin-1-yl)-trans-cyclohexylmethyl]-amino}-pyrazin-2-yl)-1,4-dihydro-2H-isoquinolin-3-one1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one,

(S)-5-(5-Chloro-1-methyl-2-oxo-1,2-dihydro-pyridin-3-yl)-6-(4-chloro-phenyl)-2-(2,4-dimethoxy-pyrimidin-5-yl)-1-isopropyl-5,6-dihydro-1H-pyrrolo[3,4-d]imidazol-4-one4-[(S)-5-(3-Chloro-2-fluoro-phenyl)-2-(2,4-dimethoxy-pyrimidin-5-yl)-3-isopropyl-6-oxo-3,4,5,6-tetrahydro-pyrrolo[3,4-d]imidazol-4-yl]-benzonitrile

(S)-5-(5-Chloro-2-oxo-1,2-dihydro-pyridin-3-yl)-6-(4-chloro-phenyl)-2-(2,4-dimethoxy-pyrimidin-5-yl)-1-isopropyl-5,6-dihydro-1H-pyrrolo[3,4-d]imidazol-4-one

(S)-5-(3-chloro-4-fluorophenyl)-6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-1-((R)-1-methoxypropan-2-yl)-5,6-dihydropyrrolo[3,4-d]imidazol-4(1H)-one,

and

(S)-5-(5-chloro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)-6-(4-chlorophenyl)-2-(2,4-dimethoxy-d6-pyrimidin-5-yl)-1-((R)-1-methoxypropan-2-yl)-5,6-dihydropyrrolo[3,4-d]imidazol-4(1H)-one.

21. The MDM2i for use in the treatment of cancer according to any one ofitems 1 to 16, or 19, the use of a MDM2i for the preparation of amedicament for the treatment of a cancer according to item 17 or 19, orthe method of treating cancer according to item 18 or 19, wherein theMDM2i is

(S)-5-(5-Chloro-1-methyl-2-oxo-1,2-dihydro-pyridin-3-yl)-6-(4-chloro-phenyl)-2-(2,4-dimethoxy-pyrimidin-5-yl)-1-isopropyl-5,6-dihydro-1H-pyrrolo[3,4-d]imidazol-4-oneor

(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazinyl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one.

22. The MDM2i for use in the treatment of cancer according to any one ofitems 1 to 16, or 19, the use of a MDM2i for the preparation of amedicament for the treatment of a cancer according to item 17 or 19, orthe method of treating cancer according to item 18 or 19, wherein theMDM2i is(S)-5-(5-Chloro-1-methyl-2-oxo-1,2-dihydro-pyridin-3-yl)-6-(4-chloro-phenyl)-2-(2,4-dimethoxy-pyrimidin-5-yl)-1-isopropyl-5,6-dihydro-1H-pyrrolo[3,4-d]imidazol-4-one.

23. The MDM2i for use in the treatment of cancer according to any one ofitems 1 to 16, or 19 to 22, the use of a MDM2i for the preparation of amedicament for the treatment of a cancer according to any one of items17 or 19 to 22, or the method of treating cancer according to item 18 or19 to 22, wherein the MDM2i for use in the treatment of cancer, the useof a MDM2i for the preparation of a medicament or the method of treatingcancer further comprise another pharmaceutical ingredient which isadministered to a patient.

24. The MDM2i for use in the treatment of cancer according to item to23, the use of a

MDM2i for the preparation of a medicament for the treatment of a canceraccording to item to 23, or the method of treating cancer according toitem to 23, wherein the another pharmaceutical ingredient is anotherantineoplastic agent.

25. The MDM2i for use in the treatment of cancer according to item to 24the use of a MDM2i for the preparation of a medicament for the treatmentof a cancer according to item to 24, or the method of treating canceraccording to item to 24, wherein more than one further antineoplasticagent is administered.

26. The MDM2i for use in the treatment of cancer according to any one ofitems 23 to 25, the use of a MDM2i for the preparation of a medicamentfor the treatment of a cancer according to any one of items 23 to 25, orthe method of treating cancer according to any one of items 23 to 25,wherein MDM2i is administered intermittently in at least threeconsecutive doses and the period between each two consecutive doses isat least 1 week.

27. The MDM2i for use in the treatment of cancer according to any one ofitems 23 to 25, the use of a MDM2i for the preparation of a medicamentfor the treatment of a cancer according to any one of items 23 to 25, orthe method of treating cancer according to any one of items 23 to 25,wherein MDM2i is administered intermittently in at least threeconsecutive doses and the period between each two consecutive doses isat least 2 weeks.

28. The MDM2i for use in the treatment of cancer according to any one ofitems 23 to 25, the use of a MDM2i for the preparation of a medicamentfor the treatment of a cancer according to any one of items 23 to 25, orthe method of treating cancer according to any one of items 23 to 25,wherein MDM2i is administered intermittently in at least threeconsecutive doses and the period between each two consecutive doses isat least 3 weeks.

29. The MDM2i for use in the treatment of cancer according to any one ofitems 1 to 16, or 19 to 28, the use of a MDM2i for the preparation of amedicament for the treatment of a cancer according to any one of items17 or 19 to 28, or the method of treating cancer according to item 18 or19 to 28, wherein the MDM2i is(S)-5-(5-Chloro-1-methyl-2-oxo-1,2-dihydro-pyridin-3-yl)-6-(4-chloro-phenyl)-2-(2,4-dimethoxy-pyrimidin-5-yl)-1-isopropyl-5,6-dihydro-1H-pyrrolo[3,4-d]imidazol-4-one.

30. The MDM2i for use in the treatment of cancer according to any one ofitems 1 to 16, or 19 to 28, the use of a MDM2i for the preparation of amedicament for the treatment of a cancer according to any one of items17 or 19 to 28, or the method of treating cancer according to item 18 or19 to 28, wherein the MDM2i is(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl|amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one.

The term “Mdm2 inhibitor” or “Mdm2i” denotes herein any compoundinhibiting the HDM-2/p53 or HDM-4/p53 interaction with an IC₅₀ of lessthan 10 μM, preferably less than 1 μM, preferably in the range of nM,measured by a Time Resolved Fluorescence Energy Transfer (TR-FRET)Assay. The inhibition of p53-Hdm2 and p53-Hdm4 interactions is measuredby time resolved fluorescence energy transfer (TR-FRET). Fluorescenceenergy transfer (or Foerster resonance energy transfer) describes anenergy transfer between donor and acceptor 5 fluorescent molecules. Forthis assay, MDM2 protein (amino acids 2-188) and MDM4 protein (aminoacids 2-185), tagged with a C-terminal Biotin moiety, are used incombination with a Europium labeled streptavidin (Perkin Elmer, Inc.,Waltham, Mass., USA) serving as the donor fluorophore. The p53 derived,Cy5 labeled peptide Cy5- TFSDLWKLL (p53 aa18-26) is the energy acceptor.Upon excitation of the donor 10 molecule at 340 nm, binding interactionbetween MDM2 or MDM4 and the p53 peptide induces energy transfer andenhanced response at the acceptor emission wavelength at 665 nm.Disruption of the formation of the p53-MDM2 or p53-MDM4 complex due toan inhibitor molecule binding to the p53 binding site of MDM2 or MDM4results in increased donor emission at 615 nm. The ratiometric FRETassay readout is calculated from the 15 raw data of the two distinctfluorescence signals measured in time resolved mode (countrate 665nm/countrate 615 nm×1000). The assay can be performed according to thefollowing procedure: The test is performed in white 1536wmicrotiterplates (Greiner Bio-One GmbH, Frickenhausen, Germany) in atotal volume of 3.1 μl by combining 100 nl of compounds diluted in 90%DMSO/10% H₂O (3.2% final DMSO concentration) with 2p1 Europium 20labeled streptavidin (final concentration 2.5 nM) in reaction buffer(PBS, 125 mM NaCl, 0.001% Novexin (consists of carbohydrate polymers(Novexin polymers), designed to increase the solubility and stability ofproteins; Novexin Ltd., ambridgeshire, United Kingdom), Gelatin 0.01%,0.2% Pluronic (block copolymer from ethylenoxide and propyleneoxide,BASF, Ludwigshafen, Germany), 1 mM DTT), followed by the addition of0.5p1 MDM2-Bio or MDM4-Bio diluted in assay buffer (final concentration10 nM). Allow the solution to pre-incubate for 15 minutes at roomtemperature, followed by addition of 0.5p1 Cy5-p53 peptide in assaybuffer (final concentration 20 nM). Incubate at room temperature for 10minutes prior to reading the plate. For measurement of samples, anAnalyst GT multimode microplate reader (Molecular Devices) with thefollowing settings 30 is used: Dichroic mirror 380 nm, Excitation 330nm, Emission Donor 615 nm and Emission Acceptor 665 nm. IC50 values arecalculated by curve fitting using XLfit. If not specified, reagents arepurchased from Sigma Chemical Co, St. Louis, Mo., USA.

According to one embodiment, a Mdm2 inhibitor can be for example acompound of any of the following formulas:

S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one

(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl|amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one

(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(6-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl|amino}-pyridin-3-yl)-1,4-dihydro-2H-isoquinolin-3-one

(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(6-{methyl-[4-(3-methyl-4-oxo-imidazolidin-1-yl)-trans-cyclohexylmethyl|amino}-pyridin-3-yl)-1,4-dihydro-2H-isoquinolin-3-one

(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(5-{methyl-[4-(3-methyl-4-oxo-imidazolidin-1-yl)-trans-cyclohexylmethyl]-amino}-pyrazin-2-yl)-1,4-dihydro-2H-isoquinolin-3-one1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl|amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one,

(S)-5-(5-Chloro-1-methyl-2-oxo-1,2-dihydro-pyridin-3-yl)-6-(4-chloro-phenyl)-2-(2,4-dimethoxy-pyrimidin-5-yl)-1-isopropyl-5,6-dihydro-1H-pyrrolo[3,4-d]imidazol-4-one4-[(S)-5-(3-Chloro-2-fluoro-phenyl)-2-(2,4-dimethoxy-pyrimidin-5-yl)-3-isopropyl-6-oxo-3,4,5,6-tetrahydro-pyrrolo[3,4-d]imidazol-4-yl]-benzonitrile

(S)-5-(5-Chloro-2-oxo-1,2-dihydro-pyridin-3-yl)-6-(4-chloro-phenyl)-2-(2,4-dimethoxy-pyrimidin-5-yl)-1-isopropyl-5,6-dihydro-1H-pyrrolo[3,4-d]imidazol-4-one

(S)-5-(3-chloro-4-fluorophenyl)-6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-1-((R)-1-methoxypropan-2-yl)-5,6-dihydropyrrolo[3,4-d]imidazol-4(1H)-one,

or

(S)-5-(5-chloro-1-methyl-2-oxo-1,2-dihydropyridin-3-yl)-6-(4-chlorophenyl)-2-(2,4-dimethoxy-d6-pyrimidin-5-yl)-1-((R)-1-methoxypropan-2-yl)-5,6-dihydropyrrolo[3,4-d]imidazol-4(1H)-one.

In a particular embodiment, the MDM2i is(S)-5-(5-Chloro-1-methyl-2-oxo-1,2-dihydro-pyridin-3-yl)-6-(4-chloro-phenyl)-2-(2,4-dimethoxy-pyrimidin-5-yl)-1-isopropyl-5,6-dihydro-1H-pyrrolo[3,4-d]imidazol-4-one(hereinafter compound A), or a pharmaceutically acceptable salt thereof.

In another embodiment, the MDM2i is(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one(hereinafter compound B).

The term “subject” or “patient” as used herein includes animals, whichare capable of suffering from or afflicted with a cancer or any disorderinvolving, directly or indirectly, a cancer. Examples of subjectsinclude mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats,cats, mice, rabbits, rats and transgenic non-human animals. In thepreferred embodiment, the subject is a human, e.g., a human sufferingfrom, at risk of suffering from, or potentially capable of sufferingfrom cancer. In a particular embodiment, subject or patient is human.

The term “treating” or “treatment” as used herein denotes to arrest,delay the onset (i.e., the period prior to clinical manifestation of adisease) and/or reduce the risk of developing or worsening a disease, orcomprises relieving, reducing or alleviating at least one symptom in asubject or effecting a delay of progression of a disease. For example,treatment can be the diminishment of one or several symptoms of adisorder or complete eradication of a disorder, such as cancer.

The term “antineoplastic agent” is a pharmaceutical active ingredientthat exhibits antiproliferative or anti-cancer activity. Possibleantineoplastic agents suitable for combination treatment include, butare not limited to BRAF inhibitors (e.g. (S)-methyl-1-(4-(3-(5-chlorofluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamateor vemurafenib); anaplastic lymphoma kinase (ALK) inhibitors (e.g.ceritinib, AE684, Alectinib, Crizotinib, AP26113, ASP3026, ADZ3463);aromatase inhibitors (e.g. atamestane, exemestane and formestane,aminoglutethimide, roglethimide, pyridoglutethimide, trilostane,testolactone, ketokonazole, vorozole, fadrozole, anastrozole orletrozole); antiestrogens (tamoxifen, fulvestrant, raloxifene orraloxifene hydrochloride); antiandrogen (e.g. bicalutamide);topoisomerase I inhibitors (e.g. topotecan, gimatecan, irinotecan,camptothecian and its analogues, 9-nitrocamptothecin and themacromolecular camptothecin conjugate PNU-166148 (compound Al inWO99/17804); topoisomerase II inhibitors (e.g. doxorubicin,daunorubicin, epirubicin, idarubicin, nemorubicin, mitoxantrone,losoxantrone, etoposide or teniposide); microtubule active compounds(e.g. paclitaxel, docetaxel, vinblastine, vinblastine sulfate,vincristine, vincristine sulfate, vinorelbine, dis-codermolides,cochicine); alkylating compounds (e.g. cyclophosphamide, ifosfamide,melphalan or nitrosourea); histone deacetylase inhibitors; compoundswhich induce cell differentiation processes; cyclooxygenase inhibitors;MMP inhibitors; mTOR inhibitors; antineoplastic antimetabolites; platincompounds; compounds targeting/decreasing a protein or lipid kinaseactivity; anti-angiogenic compounds; compounds which target, decrease orinhibit the activity of a protein or lipid phosphatase; gonadorelinagonists (e.g. abarelix, goserelin and goserelin acetate); methionineaminopeptidase inhibitors; bisphosphonates; biological responsemodifiers; antiproliferative antibodies; heparanase inhibitors;inhibitors of Ras oncogenic isoforms; telomerase inhibitors; proteasomeinhibitors; compounds used in the treatment of hematologic malignancies;compounds which target, decrease or inhibit the activity of Flt-3; Hsp90inhibitors; kinesin spindle protein inhibitors; MEK inhibitors;leucovorin; EDG binders; antileukemia compounds; ribonucleotidereductase inhibittors; S-adenosylmethionine decarboxylase inhibitors;angiostatic steroids; corticosteroids; other chemotherapeutic compounds(as defined below); photosensitizing compounds (e.g. VISUDYNE andporfimer sodium).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the PK of Compound A on SJSA-1 tumor-bearing rat afterone single intravenous (i.v.) treatment

FIG. 2 depicts the PK and PD of Compound A on SJSA-1 tumor-bearing ratafter one single i.v. treatment

FIG. 3 depicts the Tumor growth after a single i.v. treatment of SJSA-1tumor-bearing nude rat with compound A

FIG. 4 depicts the Change in body weight (BW) after a single i.v.treatment of SJSA-1 tumor-bearing nude rat with compound A

FIG. 5 depicts the Efficacy of compound A after a single i.v. treatmentof SJSA-1 tumor-bearing nude rat—individual data

FIG. 6 depicts the Bone marrow recovery after a single i.v. treatment

FIG. 7 depicts the Correlation between white blood cell count in bloodand on bone marrow section from sternum

FIG. 8A shows the tumor growth and the change in body weight of nuderats over 42 days after i.v. (q3w, i.e. once every three weeks)treatment of SJSA-1 tumor-bearing nude rat.

FIG. 8B shows the tumor growth and the change in body weight of nuderats over 42 days after i.v. (q3w, i.e. once every three weeks)treatment of SJSA-1 tumor-bearing nude rat.

FIG. 9A depicts the Effect of the i.v. treatment (q3w) on the whiteblood cells (WBC)

FIG. 9B depicts the Effect of the i.v. treatment (q3w) on neutrophils

FIG. 9C depicts the Effect of the i.v. treatment (q3w) on the plateletcount

FIG. 10A shows the tumor growth and the change in body weight of nuderats over 42 days of q3w i.v. treatment with Compound A at 13.7 and 18.2mg/kg

FIG. 10B shows the tumor growth and the change in body weight of nuderats over 42 days of q3w i.v. treatment with Compound A at 13.7 and 18.2mg/kg

FIG. 11A depicts the Effect of the i.v. treatment (q3w) with Compound Aat 13.7 and 18.2 mg/kg on the white blood cells (WBC)

FIG. 11B depicts the Effect of the i.v. treatment (q3w) with Compound Aat 13.7 and 18.2 mg/kg on the neutrophils

FIG. 11C depicts the Effect of the i.v. treatment (q3w) with Compound Aat 13.7 and 18.2 mg/kg on the platelet count

FIG. 12 depicts the PK study on SJSA-1 tumor bearing rat after onesingle treatment with compound A per os

FIG. 13 shows the drug concentration and the PD response in tumor aftersingle administration

FIG. 14 shows the tumor growth and the change in body weight of nuderats over 42 days of q3w p.o. treatment with compound A

FIG. 15A shows the white blood cells and platelets count over the 42days of q3w p.o. treatment with compound A

FIG. 15B shows the white blood cells and platelets count over the 42days of q3w p.o. treatment with compound A

FIG. 15C shows the white blood cells and platelets count over the 42days of q3w p.o. treatment with compound A

FIG. 16 depicts that the Low dose of Mdm2i does not trigger the samebiochemical effect as does a high dose

FIG. 17 depicts the Combination of an intermittent and more frequentdosing regimen of Mdm2i has having synergistic effect on efficacy

FIG. 18 depicts the Efficacy on SJSA-1 tumor bearing rat afteradministering Compound A intermittently with a high dose, daily with alow dose and combination of the both dosing schedules

FIG. 19 depicts the Tolerability on SJSA-1 tumor bearing rat afteradministering Compound A intermittently with a high dose, daily with alow dose and combination of the both dosing schedules

FIG. 20 depicts the Efficacy of Mdm2i at 27 mg/kg q3w per os in melanomaPTX bearing rat. “Cmp A” is an abbreviation for a “Compound A”.

FIG. 21 depicts the Tolerability of Mdm2i at 27 mg/kg q3w per os inmelanoma PTX bearing rat

FIG. 22 depicts the Efficacy of intermittently administered compound Ain combination with ceritinib in SHSY5Y tumor bearing mice. “Cmp A” isan abbreviation for a “Compound A”

DETAILED DESCRIPTION OF THE DISCLOSURE

Currently, Mdm2 inhibitors are dosed daily, optionally with drugholidays. The break after a series of daily treatments with Mdm2i mayhave been extended in certain cases due to tolerability issues.Exceptionally, Mdm2 inhibitors are dosed at weekly intervals. Now, itwas found that(S)-5-(5-Chloro-1-methyl-2-oxo-1,2-dihydro-pyridin-3-yl)-6-(4-chloro-phenyl)-2-(2,4-dimethoxy-pyrimidin-5-yl)-1-isopropyl-5,6-dihydro-1H-pyrrolo[3,4-d]imidazol-4-one(compound A) in a higher single dose administered intravenously or peros allowed for the first time a strong Puma mRNA induction (Emax 70 foldinduction) which was never reached previously with a lower per os (p.o.)dose of compound A or(S)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one(compound B). It is interesting to note that Mdm2, the inhibitor of p53,had the lowest mRNA induction. Such high Puma mRNA induction in thetumor was followed by a strong caspase-3 activation 24 h post-treatmentwhich translated in a dramatic decrease in tumor cell density 48 and 72h post-treatment. The strong induction of the apoptotic pathway wasclearly identified as the main driver of the striking and unexpectedtumor regression induced by a single treatment at high dose. Indeed,single i.v. treatment with compound A at 20 mg/kg induced a completeSJSA-1 tumor response (100% regression) in 82% (9/11) of treated rat for42 days. Moreover, once every 3 weeks (q3w) p.o. treatment with compoundA at 27 mg/kg induced an 88 and 27% SJSA-1 tumor regression after oneand two cycles, respectively.

We found that in order to trigger prolonged apoptosis or sustainedantiproliferative effect with strong Puma induction (i.e. at least 20fold induction of mRNA expression compared to the mRNA expression innon-treated cancer cells) compound A has to be administered at asufficiently high dose. Said dose allows the drug to be administeredintermittently without significantly losing efficacy and potentiallyimproving tolerability. Single doses of Compound A can be dosed every 2weeks. Also breaks of 3 weeks, 4 weeks, 6 weeks, or even intermittenceof 60 days can still show significant effect on the tumor. Below saidhigh dose, compound A only induces Puma mRNA expression up to about 5-6fold and as a consequence requires to be administered continuously, forexample daily, in order to attain a continuous antiproliferative effect.Once the drug has been administered long enough, even at lower dose, abreak from treatment can be made, but the treatment cycle has to berepeated in at least about 2 weeks, otherwise the antiproliferativeeffect is not observed anymore.

In one embodiment, according to the present disclosure, Mdm2i used forthe treatment of cancer is provided, wherein a single dose of the Mdm2iis to be administered at least every two weeks, and not longer thanevery 60 days. In another embodiment, the single dose of the Mdm2i is tobe administered at least every three weeks, and not longer than every 60days.

Other Mdm2i than compound A can also achieve strong Puma induction, butthe dose to be used is dependent on the compound's potency. Withoutwanting to be bound to any theory, it is believed that the dose of aMdm2i to generate a prolonged effect via very pronounced Puma inductionneeds to be lower when the Mdm2i is more potent. But in principle alsolow potent Mdm2i can activate this second level modality that leads tolong-lasting effect, if only administered at a dose that reachessufficient plasma exposure. About 26% tumor regression can be achievedif the Mdm2i is above the G180-concentration for at least 8 hours, andof more than 90% if the Mdm2i exposure persists above G180 for at least17 hours. GI-80 is the dose necessary to cause 80% of tumor cell growthinhibition. Therefore, generally, the high dose or higher dose of aMdm2i is the dose that causes the Mdm2i to persist for at least 8 hours,preferably at least 10 hours, in plasma in vivo at least at aconcentration that otherwise causes GI-80 when exposing the tumor cellsin vitro to the Mdm2i for 8 hours. GI-80 concentration can be measuredby any proliferation test. For example, CellTiter-Glo® Luminescent CellViability Assay is used. For example cells in vitro are treated with theMdm2i for 8 hours, then the cells are washed to remove the compound inthe medium and determination of the number of viable cells is made after72 hours. This is repeated with various concentrations to identify GI-80concentration. The high dose has to reach or supersede in vivo saidGI-80 concentration of the Mdm2i for at least 8 hours. Low dose is lowerthan the lowest high dose. Unfortunately, administering higher doses ofMdm2i does not always translate into sufficient plasma exposure simplydue to specific pharmacokinetics of the compound, particularly if givenorally, because for example low bioavailability can prevent the drugfrom achieving sufficiently high plasma levels. This drawback of oraladministration is overcome when the Mdm2i is administered intravenously.

The “dose” as mentioned herein in the context of an administered dosecan also mean strength.

Thus, it is one objective of this disclosure to provide a MDM2i for usein the treatment of cancer, wherein MDM2i is to be administered to asubject intermittently and the period between at least three consecutivedoses is at least 2 weeks, at least 3 weeks, at least 4 weeks, at least6 weeks or 60 days, and not longer than 60 days. MDM2i is to beadministered to a subject intermittently in at least three consecutivedoses and the period between each two consecutive doses of the threedoses is at least 2 weeks, at least 3 weeks, at least 4 weeks, at least6 weeks or 60 days. The upper limit is set based on the available data,but we allow for a possibility, that even more infrequent administrationmay lead to clinically acceptable outcome and could be useful. Toimprove patient compliance, the administration regimen for the Mdm2i canbe once every 3 weeks or 4 weeks, particularly once every 3 weeks.

We found that the problem of suboptimal exposure of the Mdm2i in a body,particularly if it has cell proliferation IC50 of more than 1 μM, can besolved by administering the drug intravenously. As an example, we foundthat lower doses of compound A (20 mg/kg) can be administeredintravenously, while 27 mg/kg were needed orally to stimulate the sameresponse. Therefore, administering Mdm2i intravenously opens a chancefor Mdm2i of a lower potency to achieve the aforementioned second stageof reactivity with extended antiproliferative effect. This way, there isa chance to dose the drug less frequently, because only intravenousadministration will lead to the required exposure. In addition,administering Mdm2i intravenously at a lower dose compared to the dosethat would be needed for oral administration can at least offer sometolerability advantage.

Therefore, in one embodiment, we provide Mdm2i for use in the treatmentof cancer, wherein MDM2i is to be administered intravenously.

In another embodiment, the intermittent dosing of a MDM2i can besupplemented by another dosing regimen of a second dose of the MDM2ithat is different to the dose used in the intermittent dosing of asingle dose. Combining the intermittent dosing schedule with anothermore frequent schedule allows reducing the dose of Mdm2i used in each ofthe schedules and thus further improves tolerability. Administering ahigh dose of a Mdm2i intermittently while also dosing the Mdm2i morefrequently, e.g. daily at a lower dose enables to reduce doses for bothschedules to the level that would otherwise not be efficacious, at leastin one of the two dosing schedules, if said dosing schedules was usedalone. Combining the treatment with a high and a low dose at differentschedules also proved to be synergistically effective. In oneembodiment, the intermittent dosing, where Mdm2i is administered atleast every 2 weeks, daily treatment of the Mdm2i can be superimposed.We found that combining two dosing regimens of compound A, namely thetreatment once every 3 weeks with a higher dose and a 2 week dailytreatment with a lower dose with a 2-week break every 28-day cycle, leadto synergistic antitumor effect of both treatments. The second dosingregimen that is added to the intermittent treatment can start on thesame, consecutive or other day. The second dosing regimen can be forexample daily, optionally with a break. The break after a series ofdaily treatments can be at least 1 day long, 2 days, 3 days, 4 days, 1week, 2 weeks, or 3 weeks and at most 26 days long. In one embodiment,the dose of the second dosing regimen is to be administered 1 to 14 daysafter the first dose has been administered. In a specific embodiment,the second dosing schedule with a lower dose of Mdm2i starts on the nextday after single high dose has been administered. The dose that isadministered daily can be administered for two weeks followed by aperiod of two weeks without treatment and then the treatment cycle canbe repeated. Generally, the dose used for intermittent dosing will behigher than the second dose used in more frequent dosing that is addedto the intermittent dosing. Mdm2i can be administered either per os orintravenously, or in combination thereof. For example, intermittent doseat least every 2, 3, 4, 6 weeks or 60 days can be administeredintravenously, whereas the second daily dose can be given orally.However, both doses can be administered intravenously, or both orally.In one embodiment, the first dose that is administered intermittentlycan be administered with periods between two consecutive administrationsof at least 2 weeks.

In one aspect, the second dosing schedule that is added to theintermittent dosing schedule can comprise administering the Mdm2i for aperiod of at least 5 days followed by a period of 1 day or more, andrepeating the cycle while the patient is treated with the Mdm2iintermittently at the different dose. However, additional second dosingschedules include, for example, cycles of 2 weeks on, 1 or 2 weeks off;3 weeks on 1, 2 or 3 weeks off; 4 weeks on 1, 2, 3 or 4 weeks off; 1week on, 3 weeks off; 3 weeks on, 1 weeks off; 4 weeks on, 1 week off.

In addition of adding a second dosing schedule to the intermittentdosing, a clinical outcome of a MDM2i treatment with the intermittentdosing can be improved by administering a further pharmaceuticalingredient to the subject. The further pharmaceutical ingredient can beanother Mdm2i, but most often it will be a drug with a differentmechanism of action. It is contemplated herein that giving anotherantineoplastic agent in addition to intermittently dosed Mdm2i canachieve improved antitumor effect. In addition, intermittent dosingopens up more flexibility to combining Mdm2i with another antineoplasticagent as by reducing the frequency of Mdm2i dosing, tolerability canimprove and thus allows more options to add another anticancer drug. Inone embodiment, the Mdm2i is administered intermittently as describedherein in combination with a BRAF inhibitor or an ALK inhibitor.Specifically, the another pharmaceutical ingredient is(S)-methyl-1-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate.In another embodiment, the combination is made with Ceritinib. Where theMdm2i is combined with another pharmaceutical ingredient, the Mdm2i canbe administered intermittently with the periods between single dosesbeing at least 1 week, at least 2 weeks, at least 3 weeks, at least 4weeks, at least 6 weeks or 60 days, and not longer than 60 days.

The present disclosure provides also compound A for use in thetreatment, wherein the compound A is administered intermittently, e.g.the period between each two doses of at least three doses is at least 1week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6weeks or 60 days, and not longer than 60 days, and(S)-methyl-1-(4-(3-(5-chloro-2-fluoro(methylsulfonamido)phenyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)propanylcarbamate or Ceritinib are also used. Particularly, the compound A isadministered as a single dose at least every week, or at least everythree weeks.

The Mdm2i and additional pharmaceutical ingredient can be applied orformulated of the separate partners with or without, preferably with,instructions for combined use or to combination products. The compoundsin the combination may thus be administered entirely separately or beentirely separate pharmaceutical dosage forms. The combination partnersmay be pharmaceutical compositions that are also sold independently ofeach other and where just instructions for their combined use areprovided in the package equipment, e.g. leaflet or the like, or in otherinformation e.g. provided to physicians and medical staff (e.g. oralcommunications, communications in writing or the like), for simultaneousor sequential use for being jointly active. The Mdm2i and another activepharmaceutical ingredient can be provided as a fixed or a non-fixedcombination of the active ingredients. The term “fixed combination”means that the active ingredients, e.g. a Mdm2 inhibitor and anantineoplastic agent, are both administered to a patient simultaneouslyin the form of a single entity or dosage. In other terms: the activeingredients are present in one dosage form, e.g. in one tablet or in onecapsule. The term “non-fixed combination” means that the activeingredients are both administered to a patient as separate entitieseither simultaneously, concurrently or sequentially with no specifictime limits, wherein such administration provides therapeuticallyeffective levels of the two compounds in the body of the patient.

The cancers treated by the use of Mdm2i as described herein includecancer such as, but not limited to, bladder, breast, brain, head andneck, liver, oral, biliary tract, acute and chronic lymphoid leukemia,acute and chronic myeloid leukemia, chronic myelomonocytic leukemia,colorectal, gastric, gastrointestinal stromal, hepatocellular, glioma,lymphoma, melanoma, multiple myeloma, myeloproliferative disease,neuroendocrine, lung, non-small cell lung, pancreatic, ovarian,prostate, renal cell, sarcoma, liposarcoma and thyroid cancer. In aspecific embodiment, the cancer is melanoma. In another embodiment thecancer is neuroblastoma. In yet another embodiment, the cancer isleukemia.

Based on the data obtained with the Compound A, and knowing also thebiochemical response of(5)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl|amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one (Compound B), we can expect that the proposed dosingregiments could be used to provide advantageous efficacy or tolerabilitywith at least the Mdm2i listed above.

Mdm2i can be delivered to the subject in a pharmaceutical composition.Oral dosage forms to be used are for example tablets, capsules, sachets,micropellets, granules or the like. The oral dosage forms can comprisein addition to the Mdm2i further conventional carriers or excipientsused for pharmaceuticals. Examples of such carriers or excipientsinclude, but are not limited to, disintegrants, binders, lubricants,glidants, stabilizers, and fillers, diluents, colorants, flavours andpreservatives. One of ordinary skill in the art may select one or moreof the aforementioned carriers with respect to the particular desiredproperties of the dosage form by routine experimentation and without anyundue burden. The amount of each carriers used may vary within rangesconventional in the art. The following references disclose techniquesand excipients used to formulate oral dosage forms. See The Handbook ofPharmaceutical Excipients, 4th edition, Rowe et al., Eds., AmericanPharmaceuticals

Association (2003); and Remington: the Science and Practice of Pharmacy,20th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2003). Thedosage forms are prepared for example by blending, granulating,compressing, compacting, filling, sieving, mixing and/or tableting.

The Mdm2i can be applied in vivo intravenously, e.g. as a solution.Generally, the dosage form would be autoclaved or sterilized by usingother process before administration. The drug can be administeredintravenously by injection or infusion. Preferably, the Mdm2i is infusedintravenously over a period of less than 3 hours, more preferably in upto 2 hours, particularly in about 1 hour.

Mdm2i can be used for preparation of a medicament, where a dosage formis prepared. The latter can be further packaged and supplemented with apatient information leaflet.

The Mdm2i is administered at the therapeutically effective amount. Theterm “a therapeutically effective amount” of the Mdm2i refers to anamount of the compound that will elicit the biological or medicalresponse of a subject, for example, ameliorate symptoms, alleviateconditions, slow or delay disease progression, slow down tumor growth,or cause tumor regression, or the like. In one embodiment atherapeutically effective amount in vivo may range depending on theroute of administration, between about 0.1-500 mg/kg, or between about1-100 mg/kg. For example, for compound A, the effective in vivo amountis between 100 and 1500 mg every three weeks, particularly between 100and 800 mg every three weeks, or between 50 and 600 mg daily, whenadministered per os. For compound B, the effective amount is between 500and 4000 mg, particularly between 1500 and 4000 mg, when administeredper os. Intravenous doses would need to be lowered accordingly.

The following Examples illustrate the present disclosure.

Method and Materials Used in Examples

CompoundA—(S)-5-(5-Chloro-1-methyl-2-oxo-1,2-dihydro-pyridin-3-yl)-6-(4-chloro-phenyl)-2-(2,4-dimethoxy-pyrimidin-5-yl)-1-isopropyl-5,6-dihydro-1H-pyrrolo[3,4-d]imidazol-4-one

CompoundB—(5)-1-(4-Chloro-phenyl)-7-isopropoxy-6-methoxy-2-(4-{methyl-[4-(4-methyl-3-oxo-piperazin-1-yl)-trans-cyclohexylmethyl]-amino}-phenyl)-1,4-dihydro-2H-isoquinolin-3-one

Cell Culture

SJSA-1 osteosarcoma cells (CRL-2098, ATCC) are wild type for p53 andamplified in Mdm2 (16.9 copies, SNP 6.0) but not in Mdm4. They werecultured in RPMI 1640 (#1-41F01-I, AMIMED) supplemented with 10% FCS(#2-01F16-I, AMIMED), 2 mM L-glutamine (#5-10K00-H, AMIMED). Cells werepassaged by washing first with Dulbecco's PBS withoutCa2+/Mg2+(#3-05F29-I, AMIMED), trypsinising cells with Trypsin 0.05% inPBS with EDTA (#5-51F00-H, AMIMED), centrifuging in the respectiveculture media, and splitting cells into fresh media at a ratio of 1:8, 2times per week.

Animals

All the nude rat (Hsd: RH-Foxi^(mu), Harlan Sprague Dawley; SF480) wereallowed to adapt for 4 days and housed in a pathogen-controlledenvironment (5 mice/Type III cage) with access to food and water adlibitum. Animals were identified with transponders. Studies described inthis report were performed according to procedures covered by permitnumber 1975 issued by the Kantonales Veterinäramt Basel-Stadt andstrictly adhered to the Eidgenössisches Tierschutzgesetz and theEidgenössische Tierschutzverordnung. All experiments were done with 4 to7 rats. Mice were used for experiments with combinations of compounds.

Tumor Model

Subcutaneous tumors were induced by concentrating 1.0×10⁷ SJSA-1 cellsin 50% Matrigel® and injecting in the right flank of Harlan nude rats.Efficacy experiment could start 14 days post cell injection. Compound Awas made-up fresh for each administration. For i.v. injection (4 ml/kg),Compound A was dissolved in 30% PEG300, 10% Solutol HS 15, 6% PluronicF68 and 54% water. For per os (p.o.) injection (5 ml/kg), Compound A wasdissolved in methylcellulose 0.5% w/V in phosphate buffer pH 6.8 50 mM.The animals were treated either at a high dose (20 mg/kg i.v. or 27mg/kg p.o.) once every 3 weeks (q3w) or at a high dose (15 mg/kg p.o.)followed 24 h later by a daily low dose treatment (3 mg/kg p.o., 2 weekson/2 weeks off).

The tumor volume (TVol) and body-weight (BVV) of the animal weremeasured three times per week allowing calculation at any particulartime-point relative to the day of initiation of treatment (day 0) of thepercentage change in TVol (Δ% TVol). Tumor response was quantified bythe change in tumor volume (endpoint minus starting value in mm³) as theT/C i.e.

$\left( {\frac{\Delta{TVol}_{drug}}{\Delta{TVol}_{vehicle}} \times 100} \right).$

In the case of a tumor regression or to assess the percentage of changein TVol, the tumor response was quantified by the percentage ofregression of the starting TVol, ie

$\left( {\frac{\Delta{TVol}_{drug}}{{TVol}_{Day0}} \times 100} \right).$

Similarly, the body-weight (BW) of the animal was measured three timesper week allowing calculation at any particular time-point relative tothe day of initiation of treatment (day 0) of the percentage change inBW (Δ% BVV).

The white blood cells (WBC), neutrophils and platelets were countedusing a Sysmex (XT-2000i). Blood was collected into commerciallyprepared EDTA coated microtubes (BD Microtainer, cat #365975).

Pharmacokinetic (PK) and Pharmacodynamic (PD)

At the times indicated, animals were anaesthetized by exposure to 2-3%v/v isofluorane in medical oxygen:

Either the animal was killed without recovering from anesthetic afterblood sampling. Blood was collected into commercially prepared EDTAcoated tubes (Milian, cat #TOM-14C) in order to extract plasma. Thetissues were excised, weighed and rapidly frozen in liquid nitrogen.

Or a tumor biopsy was collected by using a biopsy gun and flushing theneedle with RLT buffer in Barney rubble tubes (Covaris, cat #520048). Inaddition, 20 μl of blood may have been collected from the tail vein anddiluted in 20 μl of water. After recovery of anesthesia, animals weretransferred in their respective cages.

Tissue, blood and plasma samples were stored frozen at −80° C. untilanalysis.

Preparation of Tissue

Frozen tissues were cryogenic dry pulverized and biopsies were sonicatedusing the CryoPrep™ system (model CP-02) from Covaris. Morespecifically, frozen tissues were transferred to disposable tubes calledTissueTubes™, placed in the CryoPrep™ system and then pulverized usingthe appropriate impact setting. The resulting powder was collected witha spatula and weighed for further processing (mRNA purification orquantification of compound in tissues). The biopsies were flushed in aBarney rubble glass tubes with 350 μl of RLT buffer and placed in theCovaris for sonication (1 min per biopsy). The resulting lysate wastransferred into a QlAshredder (79654, Qiagen) column for RNAextraction.

Pharmacodynamic (qRT-PCR)

Total RNA was purified from cell pellets using the QlAshredder (79654,Qiagen) and RNeasy Mini Kit (74106, Qiagen) according to themanufacturer's instructions, with the exception that no DNA digestionwas performed. Total RNA was eluted with 50 μL of RNase-free water.Total RNA was quantitated using the spectrophotometer ND-1000 Nanodrop®.The qRT-PCR (Quantitative Reverse Transcriptase Polymerase ChainReaction) was set up in triplicate per sample using the One-Step RT qPCRMaster Mix Plus (RT-QPRT-032X, Eurogentec), with either control primersand primers for the target, namely TaqMan Gene Expression assays(20×probe dye FAM™ (or VIC)-TAMRA (or MGB); Applied Biosystems) listedin Table 1.

TABLE 1 Source of qRT-PCR primers TaqMan ® Gene Expression Gene SpeciesKit GUS beta Human 4310888E-1012026 Gapdh Human 4310884E-0904043 Cdkn1 a(p21) Human Hs00355782_m1 BBC3 (puma) Human Hs00248075_m1 Mdm2 HumanHs01066930_m1

Pharmacokinetic

Sample Preparation and Bioanalytical Method

Concentrations of compound A in plasma and tissues were determinedsimultaneously by an UPLC/MS-MS assay. Tissues were homogenized in anequal volume of HPLC-Water (Water for chromatography, Merck) using theFast Prep®-24 system (M. P. Biomedicals, Irvine, Calif., USA). Followingaddition of 25 μl of internal standard mixture (1 μg/ml) to analyticalaliquots (25p1) of plasma or tissues homogenate the proteins wereprecipitated by the addition of 200p1 acetonitrile. The supernatant weretransferred in a fresh vial. After evaporation to dryness the sampleswere re-dissolved in 60p1 acetonitrile/water (1/1 v/v). An aliquot (5p1)of this solution was separated on a ACQUITY UPLC BEH C18 column (Waters™1.7 μm particle size, 2.1×50 mm) with a mobile phase consisting of amixture of 0.1% formic acid in water (solvent A) and 0.1% formic acid inacetonitrile (solvent B). Gradient programming was used with a flow rateof 600p1/min. After equilibration with 95% solvent A, 5p1 of sample wasinjected. Following a latency period of 0.25 min, the sample was elutedwith a linear gradient of 5-100% solvent B over a period of 0.65 minutesfollowed by a 0.35 minutes hold. The column was prepared for the nextsample by re-equilibrating over 0.25 minutes to the starting conditions.The column eluent was directly introduced into the ion source of thetriple quadrupole mass spectrometer TQD™ (Waters Corporation, Milford,Mass., USA) controlled by Masslynx™ 4.1 software. Electrospray positiveionization (ESI+) multiple reaction monitoring was used for the MS/MSdetection of the analyte. Precursor to product ion transitions of m/z555.3—□m/z 329.2 for compound A were used. The limit of quantification(LOQ) for the compound was set to 0.7 ng/mL (CV and overall bias lessthan 30%). Regression analysis and further calculations were performedusing QuanLynx™ 4.1 (Micromass) and Excel™ 2007 (Microsoft).Concentrations of unknown samples were back-calculated based on the peakarea ratios of analyte/IS from a calibration curve constructed usingcalibration samples spiked in blank plasma or tissue obtained fromanimals treated with vehicle.

Calculation of the Pharmacokinetic Parameters

Areas under the plasma concentration versus time curves (AUC) werecalculated from the mean values with linear trapezoidal rule, andfurther relevant parameters by using a non-compartmental model forextravascular dosing (WinNonlin® Professional Version 5.2, Pharsightcorp., CA, US).

Immuno-Histochemistry

All tissues were processed to FFPE according to routine procedures andfollowing fixation, rat sternum was decalcified in Citrate/EDTA bufferfor 5 days, with buffer exchange every 24 h. Sections were cut at 3 μmusing a microtome. p21 and cleaved Caspase-3 immunohistochemistry wasperformed on a Ventana Discovery XT automated immunostainer using theOmniMap anti Mouse or Rabbit HRP secondary reagent and the ChromoMap DABchromogen system (Ventana/Roche Diagnostics GmbH, Mannheim, Germany).Antigen retrieval was done by using Cell Conditioning Discovery CC1(Ventana/Roche Diagnostics) at mild (95° 8 min+100° 20 min, for cleavedCaspase-3) or standard (95° 8 min+100° 36 min, for p21) conditions. Theprimary antibody was applied manually at the desired dilution in Dakoantibody diluent, followed by incubation for 1 hour at room temperature.Corresponding negative controls were incubated with AbD only.Counterstaining of sections was done using hematoxylin (Ventana/RocheDiagnostics). After the automated staining run, slides were dehydratedin a graded series of ethanol, cleared in xylene and mounted with Pertexmounting medium.

Primary antibodies used for immunohistochemistry are described in Table2.

TABLE 2 Antibodies used for immunohistochemistry Dilution AntibodiesSpecies Clone References range Mdm2 Mouse SMP14 SC, cat. 965 1/200 mAbp21 Mouse SX118 Dako, cat. M7202 1/50 mAb p21 Mouse F-5 SC, cat. 62461/50 mAb Cleaved Rabbit — CST, cat. 9661 1/2000 Caspase-3 polyclonal(Lot #37) Ab Cleaved Rabbit 5A1E CST, cat. 9664 1/200 Caspase-3 mAb

This table shows the source of the antibodies used forimmunohistochemistry, as well as their dilution.

mRNA In Situ Hybridization

In situ hybridization was performed using the QuantiGene ViewRNA FFPEAssay kit (Affymetrix/Panomics) following the manufacturer's protocol.Gene-specific probe sets for rat Ubc (Ubiquitin C) and Bbc3 (PUMA) mRNAswere custom-designed and synthesized by Affymetrix. Bbc3 probes wereused in type 1/Fast Red and Ubc probes were used in type 6/Fast Blue.Slides were processed strictly following the QuantiGene protocol.Pre-hybridization conditions were found to be optimal with 10 min ofboiling in pre-treatment solution (Affymetrix) and 10 min of Protease QF(Affymetrix) digestion at 40° C. Briefly, five micrometer sections werecut, fixed in 10% formaldehyde, deparaffinized and rehydrated. In orderto increase accessibility to mRNAs, slides were then boiled inpre-treatment solution (Affymetrix) and digested with protease QF(Affymetrix) at optimal conditions. Sections were then hybridized for 3h at 40° C. with custom-designed QuantiGene ViewRNA probes against Bbc3and the control gene Ubc. A no-probe sample was utilized as a negativecontrol per the Affymetrix manual's recommendations. Afterhybridization, unbound probes were then flushed out with wash Buffer(Affymetrix) whereas bound probes were then amplified per protocol fromAffymetrix (branched DNA amplification) using PreAmp (25 mn at 40° C.),then Amp molecules (15 mn at 40° C.) and finally multiple Label Probeoligonucleotides conjugated to alkaline phosphatase (LP-AP) for 15 mn at40° C. LP-AP type 6 probe detection of signal was done with Fast Bluesubstrate (blue dots, Cy5 fluorescence) for 30 mn at RT in the dark,followed by LP-AP type 1 probe detection of signal with Fast RedSubstrate (red dots, Cy3 fluorescence) for 30 mn at 40° C. After signaldetection, slides were then counterstained with Mayer's haematoxylin,rinsed and mounted/coversliped by using Ultramount aqueous mountingmedium (DAKO). Images were taken with an Olympus BX51 microscopeequipped with a ColorViewIII color camera (Soft Imaging Sytem).

Probes used for mRNA ISH are described in Table 3.

TABLE 3 Probes used for mRNA ISH Probes References Rat Ubc (Ubiquitin C)Affimetrix, cat. VC6-10047-1 Rat Bbc3 (PUMA) Affimetrix, cat.VC1-13801-1

Example 1

Pharmacokinetics (PK) of Compound a after Single i.v. Injection at 20mg/kg

FIG. 1 shows Compound A concentration in plasma, tumor and liver over144 hours after one single i.v. injection. The Tmax for the compound was5 min in plasma and liver and 1 h in tumor. Compound A had a two timeshigher exposure in tumor (AUC_(0-144h)dn=16.5 h.nmol/g) compared toplasma (AUC_(0-144h) dn=8.1 h. μM). FIG. 2 shows the Compound Aconcentration in tumor and the pharmacodynamics (PD) response in tumor.Puma and p21 had a very similar mRNA induction reaching an expressionmax of 180 and 200-fold 24 h post treatment, respectively.

Example 2

PK, PD, Efficacy and Tolerability of Compound a (i.v., Once) on SJSA-1Tumor-Bearing Rat

FIG. 3 and FIG. 4 , respectively show the tumor growth and the change inbody weight of nude rats over 42 days. The single i.v. treatment withcompound A at 20 mg/kg (the higher dose) induced 92% tumor regression 14days post treatment. One rat had to be sacrificed on day 9 posttreatment because of excessive body weight (BW) loss. In spite of aslight decrease in BW 3 days post treatment for all others rats, theyrecovered quickly and gained BW during the entire experiment. Only 2 of11 tumors had an incomplete response and regrew (FIG. 5 ). These 2animals were retreated i.v. at 15 mg/kg and the tumors were stillsensitive. However, tumor regression was attenuated which could be dueto the larger tumor size. After the first treatment, p21 and Puma mRNAexpression in tumor reached a more than 50-fold increase in mRNAexpression. Mdm2 had a much lower mRNA induction (Emax=10-fold). On day59 post first treatment, all rats were treated i.v. at 20 mg/kg in orderto assess the effect of the drug on the host. The exposure of compound Awas similar in heart, jejunum, spleen, liver and bone marrow but twiceas high as in plasma (Cmax not known). The maximum increase in p21, Mdm2and cleaved Caspase-3 in jejunum and bone marrow (sternum) was alwaysobserved 3 h post treatment. All staining were back to baseline 7 dayspost treatment. The increase in cleaved caspase-3 on jejunum sectionshowed a strong correlation with the Puma (Bbc3) mRNA induction detectedby mRNA ISH. Indeed, the maximum increase in Puma was observed 3 h postlast treatment with a return to baseline 7 days post treatment. RNA ISHclearly showed that only crypt cells were stained on jejunum section.The same was true in spleen and heart.

Finally, severe bone marrow depletion could be observed 14 days posttreatment with partial recovery on day 22 (FIG. 6 ). The white bloodcell count showed that it significantly correlated with the bone marrowdepletion (R²=0.59, P=0.006, FIG. 7 ). This indicates the intermittentdosing can improve tolerability due to allowing bone marrow to recover.

Example 3

PK, PD, Efficacy and Tolerability of Compound a (i.v., q3w) on SJSA-1Tumor-Bearing Rat

FIG. 8A and FIG. 8B shows the tumor growth and the change in body weightof nude rats over 42 days. Three weeks post first treatment at 20 mg/kg,compound A induced 6% tumor regression on average. However, individualdata show that 3 rats had complete response (100% regression), 2 hadpartial response (more than 50% regression), 1 had stable disease and 1had progressive disease in spite of an early 80% regression 1 week posttreatment. After the second treatment, Compound A induced 100% tumorregression but only 2/7 animals survived the 2 full cycles. Indeed, 5rats had to be sacrificed after the second treatment because ofexcessive BW loss: the first one 10 days post second treatment and thefour others 8 days later. FIG. 9A, FIG. 9B and FIG. 9C shows the whiteblood cells (WBC), neutrophils and platelets count over the 42 days ofexperiment. Compound A induced a dramatic decrease in WBCs, neutrophilsand platelets after the first treatment and most rats only partiallyrecovered on day 21 post treatment. As a consequence, the secondtreatment brought the WBCs, neutrophils and platelets to an extremelylow level close to 0.

Example 4

PK, PD, Efficacy and Tolerability of Compound a (i.v., q3w) on SJSA-1Tumor-Bearing Rat

FIG. 10A and FIG. 10B shows the tumor growth and the change in bodyweight of nude rats over 42 days. The treatment with Compound A at 13.7and 18.2 mg/kg could induce a 66 and 88% tumor regression in average oneweek post treatment. After two weeks, all the tumors treated at 13.7mg/kg were re-growing and we decided to stop this treatment group. Threeweeks post treatment at 18.2 mg/kg, the tumors were still regressing by36% with 2 complete responses. The effect on the tumor growth tended tobe less after the second treatment as on average, the tumors wereprogressing by 118% (Table 4) and the two same tumors had completeresponses. As a consequence, the second treatment did not increase thenumber of complete responses. In terms of tolerability, the rats had aslight loss in BW 3 days post treatment, but they recovered quickly andhad gained BW by the following treatment. Actually, only one rat had adramatic loss in BW during the last day of the experiment and it cannotbe determined if it was treatment related. FIG. 11A, FIG. 11B and FIG.11C show the white blood cells, neutrophils and platelets count over the42 days of experiment. Compound A induced a strong decrease in WBCs,neutrophils and platelets after the first treatment but all measuredrats fully recovered on day 21 post treatment. The second treatment hada similar effect on cells count but rats only partially recovered on day21 post second treatment.

TABLE 4 Efficacy and tolerability of Compound A after i.v. treatment(q3w) of SJSA-1 tumor-bearing nude rat (SF480) Treatment Tumor Hosti.v., q3w 13.7 mg/kg 18.2 mg/kg 13.7 mg/kg 18.2 mg/kg Week post ΔTvolΔTvol ΔBW ΔBW treatment (%) CR (%) CR (%) Survival (%) Survival 1 −66 ±9  1/6 −80 ± 2   0/5 2.7 ± 0.9 6/6  2.5 ± 2.6 5/5 2   4 ± 37 0/6 −79 ±11  2/5 7.5 ± 1.0 6/6  9.1 ± 1.9 5/5 3 — — −36 ± 37  2/5 — — 11.9 ± 2.75/5 5 — — 22 ± 63 2/5 — — 10.6 ± 2.3 5/5 6 — — 118 ± 104 2/5 — —  5.9 ±7.9 5/5

Example 5

PK and PD with Compound a on SJSA-1 Tumor Bearing Rat after Single OralAdministration (27 mg/kg)

FIG. 12 shows the drug concentration in plasma, tumor and liver over 144hours after one single oral injection of Compound A. The Tmax for thecompound was 3 hours (h) in all matrices. Compound A showed a higherexposure in tumor (AUC_(0-144h)=277.7 h.nmol/g) in comparison to plasma(AUC_(0-144h)=111.5 h. μM). FIG. 13 shows the drug concentration and thePD response in tumor. Puma and p21 had a similar mRNA induction reachingan Emax of 162 and 180-fold 48 h post treatment. At such p.o. dose, Mdm2had a much lower Emax (34-fold).

Example 6

Efficacy on SJSA-1 Tumor Bearing Rat with a 3qw Dosing Regimen (p.o.)

FIG. 14 shows the tumor growth and the change in body weight of nuderats over 42 days of q3w p.o. treatment with compound A. Three weekspost first treatment, both doses could induce a tumor regression (27 and88% for the doses 20 and 27 mg/kg respectively). However the effect onthe tumor growth tended to be mitigated after the second treatment asonly the highest dose could still induce a tumor regression (27% at 27mg/kg). After the first treatment, Cmax and AUC_(0-24h) of compound A inblood and p21 and Puma mRNA expression in tumor nicely anddose-dependently increased. Both doses induced a slight decrease inbodyweight (BW) 3 days post each treatment but all animals recoveredquickly and had a gain in BW 3 weeks post treatment. FIG. 15A, FIG. 15Band FIG. 15C shows the white blood cells and platelets count over the 42days of experiment. Compound A induced a dose-dependent decrease inWBCs, neutrophils and platelets. WBCs and platelets fully recoveredbefore the second treatment at 20 mg/kg. For the treatment at 27 mg/kg,platelets also fully recovered but WBCs only partially.

Example 7

Effect of a Low Dose Versus High Dose

Experiments were done to evaluate the response of a high dose and a lowdose. Animals were treated with low dose of 5 mg/kg p.o. or a high doseof 27 mg/kg p.o. or 20 mg/kg i.v. Low dose of Mdm2i does not trigger thesame biochemical effect as does the high dose (FIG. 16 ).

Example 8

Combination of High and Low Dose Treatment is Highly Synergistic

The experiments were repeated on SJSA-1 tumour bearing rats withcombining two dosing schedules of compound A, one intermittent (15 mg/kgonce) and the daily dosing (1.5 mg/kg). We show on FIG. 17 thatcombining the two dosing regimens has a highly synergistic effect. Thisis a schematic presentation of multiple experiments. Further clearsynergism can be seen also on FIGS. 18 and 19 . Efficacy (FIG. 18 ) onSJSA-1 tumour bearing rat and tolerability (FIG. 19 ) was further testedwith other doses and different dosing schedules (15 mg/kg q4w (day 0)+3mg/kg q24 h 2w on/2w off (day 1); 21 mg/kg q4w (day 0)+1.5 mg/kg q24 h3w on/1w off (day 1). Combining the dosing schedules was shown toimprove efficacy and may increase tolerability, particularly as lowerdoses can be used to still achieve better tumour shrinkage.

Example 9

Efficacy and Tolerability in Melanoma Patient Derived Xenograft (PDX)Bearing Rat (Per os) The same experiments were repeated with melanomaPTX bearing rat. Efficacy (FIG. 20 ) and tolerability (FIG. 21 ) ofcompound A was tested at 27 mg/kg q3w. The intermittent dosing showedefficacy also in melanoma models.

Example 10

Efficacy of Intermittently Administered Compound a in Combination withCeritinib in SHSY5Y Tumor Bearing Mice

Similar experiments were conducted with administering a combination ofCeritinib and compound A to mice. The experiments showed (FIG. 22 ) thatCompound A can be dosed every week when combined with another compound.Ceritinib with Compound A weekly at 120 mg/kg (40 mg/kg×3 every 3 hours)resulted in better anti-tumor effect compared to ceritinib alone orcompared to combination of ceritinib+Compound A daily at 20 mg/kg (n=5).Mice have different pharmacokinetic than rats. Therefore, the dose hadto be administered 3 times every 3 hours to achieve the requiredexposures. Taking this specificity of the mice model into account,particularly a much higher clearance, the experiments on mice can beextrapolated to rats and other subjects and it is believed that the micemodel proves that at least the same effect could be achieved in rats orother subjects, particularly human, even if compound A were to beadministered at least every three weeks.

1-28. (canceled)
 29. A method of treating cancer in a subject in needthereof, comprising administering a MDM2 inhibitor to the subject for atleast three consecutive doses, wherein the period between eachconsecutive dose is at least 3 weeks and not longer than 60 days. 30.The method of claim 29, wherein the period between each consecutive doseis 3 weeks.
 31. The method according to claim 29, wherein at least onedose of the MDM2 inhibitor is administered orally.
 32. The methodaccording to claim 29, wherein each dose of the MDM2 inhibitor isadministered orally.
 33. The method according to claim 29, wherein thecancer is amplified in MDM2.
 34. The method according to claim 29,wherein the cancer is a sarcoma.
 35. The method according to claim 29,wherein the cancer is liposarcoma.
 36. The method according to claim 29,wherein the cancer is biliary tract cancer.
 37. The method according toclaim 29, wherein the subject is a human.
 38. The method according toclaim 29, comprising administering another pharmaceutical ingredient tothe subject.
 39. The method according to claim to 38, wherein thepharmaceutical ingredient is an antineoplastic agent.
 40. The methodaccording to claim 29, wherein each dose causes the MDM2 inhibitor topersist for at least 8 hours in plasma in vivo at least at aconcentration that otherwise causes 80% tumour cell growth inhibitionfollowing exposure of the tumor cells in vitro to the MDM2 inhibitor for8 hours, as measured by a proliferation test.